대사흐름분석을 통한 숙신산 생산 재조합 대장균의 대사산물 축적 예측

홍순호, 이상엽

대전광역시 유성구 구성동 373-1 한국과학기술원 화학공학과 및 생물공정연구센터

Prediction of intracellular metabolites accumulation in succinic acid producing recombinant Escherichia coli by metabolic flux analysis

Soon Ho Hong and Sang Yup Lee

Department of Chemical Engineering and BioProcess Engineering Research Center, KAIST 373-1 Kusong-dong, Yusong-gu, Taejon 305-701

Introduction

Succinic acid is a member of the C4-dicarboxylic acid family and can be used as a precursor of numerous products including pharmaceuticals, fine chemicals and biodegradable polymers. Succinic acid has been produced by chemical processes. Recently, much effort is being exerted for the production of succinic acid by microbial fermentation using renewable feedstocks, because of pollution problems associated with chemical processes.

Metabolic engineering has become a new paradigm for the more efficient production of desired bioproducts (Lee and Papoutsakis, 1999). Metabolic engineering can be defined as directed modification of cellular metabolism and properties through the introduction, deletion, and modification of metabolic pathways by using recombinant DNA and other molecular biological tools (Bailey, 1991; Lee and Papoutsakis, 1999). Recently, several metabolic engineering strategies were developed for the production of succinic acid by E. coli. In E. coli, PEP is converted to malic acid in two reaction steps catalyzed by Ppc and malate dehydrogenase (Mdh). Malic acid can also be synthesized from pyruvate by the action of malic enzyme (coded by sfcA gene). By this route the ATP released by conversion of PEP to pyruvate is conserved, and therefore, the route is energetically favorable (Stols and Donnelly, 1997). Unfortunately, the reversible reaction from pyruvate to malic acid is strongly shifted towards pyruvate. For a normal E. coli strain, the Km value of malic enzyme is 16 mM for pyruvate and only 0.4 mM for malic acid, and therefore, succinic acid is not produced from pyruvate. In an engineered strain where lactate dehydrogenase (Ldh) and pyruvate formate-lyase (Pfl) are blocked, the pool of pyruvate may, however, be sufficiently large to overcome the unfavorable equilibrium of the malic enzyme catalyzed reaction. Another obvious advantage of employing this mutant strain is reduced production of acetic acid and lactic acid.

In this study, we used a metabolically engineered E. coli strain for the production of succinic acid from glucose via pyruvate and malic acid. We also carried out metabolic flux analysis to examine the activity of other pathways in the overall metabolic networks.

Materials and methods

Metabolic flux analyses were carried out for the calculation of volumetric rates of formation of intracellular metabolites (Lee and Papoutsakis, 1999; Nielsen and Villadsen, 1994). Metabolic flux analysis is based on the pseudo-steady state assumption, which means that there is no accumulation of any intermediates.

r = TTv

The vector r is defined as the net formation rates of metabolites (mM/g DCW/h), and v is the internal reaction rates (mM/g DCW/h). T is the total stoichiometric matrix for all reactants and products of reactions. Two different metabolic flux analysis techniques were used: the conventional method and a new method in which the fluxes to the intracellular metabolite pools were introduced (see below).

Results and discussion

The result of fed-batch cultivation was analyzed by two metabolic flux analysis techniques. One is the conventional method (Lee and Papoutsakis, 1999; Nielsen and Villadsen, 1994) and the other is a new method in which the fluxes to the intracellular pyruvate and succinic acid pools were introduced. In conventional metabolic flux analysis, the metabolic network consisted of 13 reactions and 7 metabolic intermediates. Five reaction rates (glucose, succinic acid, ext-Mal, acetic acid, and biomass) and maintenance energy were used to calculate volumetric formation rates of intracellular metabolites. The biomass composition was assumed to be CH1.8O0.5N0.2 which is the average biomass composition of microorganisms (Ingraham et al., 1983). It was also assumed that 1% of biomass was regenerated in one hour at 30oC (Ingraham et al., 1983). The redundant information (lactic acid, formic acid, and ethanol formation rates) was used to check the validity of the system by comparing the calculated formation rates with the measured ones. The flux analysis seemed to have failed because the lactic acid excretion rate was considerably overestimated (230% of the measured one). Comparison of the results of metabolic flux analysis in each phase (data not shown) suggested that the overestimation of lactic acid excretion rate was due to the intracellular accumulation of pyruvate and succinic acid.

As mentioned earlier, intracellular pyruvate accumulation is necessary to change the direction of reaction catalyzed by malic enzyme (please note again that the Km values of malic enzyme are 16 mM for pyruvate and 0.26 mM for malic acid (Stols and Donnelly, 1997)). This means that the intracellular pyruvate concentration, which is initially low, needs to be increased to a certain level. To see if this really happens in our engineered strain, intracellular concentrations of pyruvate, malic acid and succinic acid were measured from the sample taken at 24, 58, 72, 86 and 103 h (Fig. 1). From the results, it was found that significant amount of intracellular pyruvate and succinic acid were accumulated. Consequently, the flux to the intracellular pools of pyruvate and succinic acid were introduced to mimic the intracellular accumulation of pyruvate and succinic acid. In this new metabolic network, there are 16 reactions and 8 metabolic intermediates. Seven reaction rates (glucose, ext-Suc, ext-Mal, acetic acid, lactic acid, formic acid, and biomass) and maintenance energy were used to calculate volumetric formation rates of intracellular metabolites.

The time profiles of the intracellularly accumulated metabolite concentrations calculated by the new flux analysis technique were compared with those of measured intracellular metabolites to check the appropriateness of introducing intracellular pyruvate and succinic acid pools (Fig. 1). The measured intracellular pyruvate concentration was maintained at a low level (below 1 mM) throughout the anaerobic cultivation period except at 86 h (5.6 mM). It was in accordance with the result of metabolic flux analysis, which predicted little intracellular accumulation of pyruvate except for Period III, in which glucose consumption rate sharply increased. This sharp increase seems to be due to the unbalance of pyruvate metabolism. Pyruvate catabolic pathways such as TCA cycle, lactic acid pathway and acetic acid pathway were mostly inactive in our system, while glucose consumption rate greatly increased in Period III most likely due to the induction of higher affinity sugar transport system. This seems to be why the intracellular pyruvate concentration increased to balance the carbon flow.

In this paper, we reported that succinic acid can be efficiently produced by employing metabolically engineered E. coli with amplified malic enzyme activities. We also introduced a new concept of introducing fluxes to the intracellular metabolite pools in the flux analysis procedure. By doing this, the concentrations of intracellular metabolites were predicted, and the differences between the measured and calculated reaction rates could be considerably reduced. This technique should be useful for analyzing the system in which some intracellular metabolites can potentially be accumulated.

Figure 1.Time profiles of calculated intracellular pyruvate (mM, ●) and succinic acid (mM, ○) concentration calculated by the new flux analysis method, and measured intracellular pyruvate (mM, ▼) and succinic acid (mM, ▽) concentration.

Acknowledgement

E. coli strain NZN111 was provided by Dr. David Clark. This work was supported by National Research Laboratory program of Ministry of Science and Technology(MOST).

Referances

1. Bailey, J. E., "Towards a science of metabolic engineering", Science, 252, 1668-1674 (1991).

2. Ingraham, J. L., Maaloe, O., Neidhardt. "Growth of the bacterial cell", Sinauer Associate, Messachusetts (1983).

3. Lee, S. Y., Papoutsakis, E. T., "Metabolic Engineering", Marcel Dekker, New York (1999).

4. Nielsen, J., Villadsen, J., "Bioreaction engineering principles", Plenum press, New York (1994).

5. Stols, L., Donnelly, M. I,. "Production of succinic acid through overexpression of NAD+-dependent malic enzyme in an Escherichia coli mutant", Appl. Environ. Microbiol., 63(7), 2695-2701 (1997)